Bax-interacting factor-1 (Bif-1) was initially identified as a protein that interacts with the pro-apoptotic protein Bax and has since been implicated in mitochondrial dynamics, autophagy and apoptosis in non-neuronal cells with its overall activity being pro-apoptotic. We recently reported the identification of novel neuron specific splice forms, Bif-1b and Bif-1c that have neuroprotective action. In contrast, Bif-1a, the only splice form expressed by non-neuronal cells, promotes apoptosis through interactions with the pro-apoptotic protein Bax. We reported reduced Bif-1b/c specifically in CNS regions predominantly affected by AD pathology, in brain regions adjacent to stroke-induced infarcts and also in aging brain. Interestingly, loss of Bif-1 alone is sufficient to cause age dependent behavioral deficits and synaptic degeneration in the mouse hippocampus. These findings suggest that the loss of Bif-1b/c may be instrumental in sensitizing neurons to a multitude of stresses associated with brain injury and disease. During the course of preliminary studies to identify and characterize Bif-1 interacting proteins in primary cortical neurons in culture we observed that protein crosslinking followed by immunoprecipitation of Bif-1a, Bif-1b and Bif-1c brought down a pro-survival member of the Bcl-2 family, Bcl-xL and mitochondrial fission factor (Mff). Bcl-xL represents the principal anti-apoptotic protein expressed in adult brain. Importantly, Bcl-xL maintains mitochondrial function and regulates neuronal metabolism by enhancing the efficiency of ATP production in mitochondria. Mff is a mitochondrial surface receptor for the fission promoting GTPase Drp1. In new preliminary studies with primary cortical neurons, in contrast to our previous studies with SH-SY5Y cells, only neuron-specific forms of Bif-1 interacted strongly with Bcl-xL and Mff, consistent with our previous studies showing that only Bif-1b and Bif-1c exhibit neuroprotective activity and promote mitochondrial elongation. These findings suggest that different Bif-1 isoforms may be endowed with distinct functions by virtue of having unique binding partners. Since little is known regarding the mechanism by which Bif-1 promotes neuronal viability, characterization of the Bif-1 interactome could provide additional clues to dissecting Bif-1?s novel function and mechanism of action in neurons. Here we propose to: i) use an integrated crosslinking and mass spectrometry approach to characterize and compare the interactome for neuron-specific and ubiquitous Bif-1 isoforms under normal and stressful conditions. This approach will allow us to extract isoform-specific differences in the Bif-1 interactome to identify selective interactions that are potentially responsible for the neuroprotective potency of neuron-specific Bif-1 isoforms and ii) validate the biological significance of interactions between Bif-1 and Bcl-xL and Mff. These studies will help elucidate some of the specific mechanisms by which alterations in the Bif-1 protein impact neuronal function and viability. Our long term goal is to identify inhibitor peptides and agonist/mimetic peptides for modulating functional interactions between Bif-1 and Bcl-xL or Mff to enhance brain function after injury and in CNS disease.